pK-matched running buffers for gel electrophoresis

ABSTRACT

pK-matched buffers, each containing two effective buffering components: one weak base and one weak acid which have similar pK a  at 25° C. (within 0.3 pK units). On agarose gels, the buffers in various concentrations were tested for separation of double-stranded DNA fragments with various DNA markers, agarose gel concentrations, and field strengths. Mobility was inversely proportional to the logarithm of molecular weight. The buffers provided high resolution without smearing at more dilute concentration than is possible with standard TAE (Tris/Acetate, pH 8.0) or TBE (Tris/Borate, pH 8.3) buffers. The buffers were also tested in 7M urea denaturing LongRanger™ sequencing gels and in non-denaturing polyacrylamide SSCP gels. The pK-matched buffers provide good separation and high resolution, at a broad range of potential pH values. In comparison to TAE and TBE, pK-matched buffers provide higher voltage and current stability, lower working concentration, more concentrated stock solutions, and lower current per unit voltage, resulting in less heat generation.

[0001] This application claims the benefit of U.S. application Ser. No.09/536,392 filed Mar. 28, 2000, which claims the benefit of U.S.Provisional Application Serial No. 60/127,087, filed Mar. 31, 1999.

GOVERNMENT RIGHTS

[0002] This invention was made with partial funding under Grant RO1-HL39762 from the National Institutes of Health of the United States. TheUnited States government has certain rights in the invention.

BACKGROUND

[0003] Since Tiselius pioneered electrophoretic separation of humanserum albumin, a-, β- and γ-globulin in 1937, electrophoresis ofbiological molecules has been critical to biomedical research (1).Electrophoretic analysis has become more sophisticated, specialized anduseful as new types of electrophoresis are developed (2,3). McDonell etal. and Southern offered detailed descriptions of standard agarose gelelectrophoresis and its use for DNA analysis (4,5). Pulsed-field agarosegel electrophoresis is an alternative for separation of very large DNAfragments up to 2000 kb (6). Another important application ispolyacrylamide gel electrophoresis for separation of small DNA segments,such as dideoxy sequencing analysis (7,8) and SSCP analysis (9-11).

[0004] Electrophoresis of nucleic acids in agarose and polyacrylamidegels is generally performed with TAE or TBE buffers. These buffersperform well in many applications, but certain limitations exist. A keylimitation is buffering capacity which determines the workingconcentration and, in turn, determines the rate at which electrophoresiscan occur without distortions due to heating. Limiting buffer capacitymay require a change of buffer when long electrophoresis times arerequired, e.g., in mutation scanning using restriction endonucleasefingerprinting or SSCP. TAE buffer cannot be used for sequencing gelsbecause of its low buffering capacity and TAE has a relatively lowsolubility, such that the maximal stock solution is 20× (2,3).Typically, laboratories that perform sequencing or SSCP-type mutationscanning prepare large volumes of stock solution.

SUMMARY OF THE INVENTION

[0005] This invention provides pK-matched buffers comprising a mixtureof a weak acid and a weak base which have pKa values at 25° C. withinabout 0.3 units of one another. The buffers are useful as runningbuffers for gel electrophoresis of nucleic acids or polypeptides. ThesepK-matched buffers have the following advantages relative to standardTBE and TAE electrophoresis buffers: 1) high resolution, 2) highelectrophoretic stability, 3) low working concentration, and 4) a widerange of pH values for selection.

[0006] The invention includes an improved gel electrophoresis method ofseparating nucleic acids or polypeptides. The improvement comprisesrunning the electrophoresis in a pK-matched buffer of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0007]FIG. 1A is a diagram illustrating the principle of pK-matchedbuffers, using TRI/TRI buffer as an example.

[0008]FIG. 1B is a comparison of electrophorectic stability betweenpK-matched buffer ETH/CAP and standard TBE buffer.

[0009]FIGS. 2A, 2B and 2C are photographs showing the effects ofconcentration of three pK-matched buffers, TRI/TRI, ETH/CAP, andBIS/ACE. FIGS. 2D and 2E are photographs showing the effects ofconcentration of standard TBE and TAE buffers.

[0010] FIGS. 3A-3C are photographs showing the effects of agaroseconcentration and applied voltage using pK-matched buffers TRI/TRI,ETH/CAP, and BIS/ACE and standard buffers TBE and TAE.

[0011] FIGS. 4A-4D are autoradiographs showing the effects of the pKmatched buffers TRI/TRI, ETH/CAP, and BIS/ACE and standard buffer TBE inSanger dideoxy sequencing analysis.

[0012]FIGS. 5A and 5B are autoradiographs showing the effects ofpK-matched buffer TRI/TRI and standard buffer TBE in didoxyfingerprinting (ddF).

DETAILED DESCRIPTION OF THE INVENTION

[0013]FIG. 1A illustrates the principle of pK-matched buffers, usingTRI/TRI buffer as an example. TRI/TRI is a mixture of Triethanolamineand TRICINE. TRICINE is (N-tris[hydroxymethyl]methylglycine. The ionsflow between anode and cathode under an electric field strength at arate which depends on the net charge and the size of the ion. Thedissociated, negatively charged TRICINE⁻ flows from cathode to anode,gives out an electron, and changes into its neutral form. Thedissociated, positively charged Triethanolamine⁺ flows from anode tocathode, accepts an electron, and changes into its neutral from. Thechemical flows happen for balance. Ideally, the flow rates of the twoions are equal and the reservoirs have the same volume. For example, atthe anode, due to increase the undissociated TRICINE concentration anddecrease in dissociated Triethanolamine⁺ concentration, chemicalbalances occur among [TRICINE⁻], [TRICINE], [Triethanolamine⁺] and[Triethanolamine], resulting in stable pH and high buffering capacity.The pH may be calculated from an equation: pH=½×(pK_(a)+pK_(a)′)+½×lg{([TRICINE⁻]×[Triethanolamine])/([TRICINE]×[Triethanolamine⁺])}. It ismodifed from Henderson-Hasselbalch equation: pH=pK_(a)+lg([TRICINE⁻]/[TRICINE]), or pH=pK_(a)′+lg([Triethanolamine]/[Triethanolamine⁺]).

[0014] A series of novel pK-matched buffers were developed andextensively tested as electrophoresis running buffers on agarose gel,denaturing sequencing gel and non-denaturing polyacrylamide gel. Theseexperiments are described below.

Materials and Methods

[0015] Reagents

[0016] Agarose LE and Seakem^(R)GTG^(R) agarose were purchased fromBoehringer Mannheim and Intermountain scientific. LongRanger™ gel wasfrom J. T. Baker. Urea and Tris (Tris[hydroxymethyl]aminomethane;C₄H₁₁NO₃; pK_(a)=8.3 at 25° C.; useful pH range 7.0-9.0; FW 121.1) werefrom Boehringer Mannheim, and Borate (H₃BO₃; pK_(a)=9.24 at 25° C.;pK_(a1)=9.14, pK_(a2)=12.74, pK_(a3)=13.80 at 20° C.; FW 61.83) andacetic acid, glacial (C₂H₃O₂; pK_(a)=4.74 at 25° C.; FW 60.05) from J.T. Baker. The other buffer components were from Sigma. Triethanolamine(2,2′,2″-Nitrilotriethanol; C₆H₁₅NO₃; pK_(a)=7.8 at 25° C.; useful pHrange 7.3-8.3; FW 149.2), TRICINE (N-tris[Hydroxymethyl]methylglycine;N-[2-Hydroxy-1,1-bis (hydroxymethyl)ethyl]glycine; C₆H₁₃NO₅; pK_(a)=8.1at 25° C.; useful pH range 7.4-8.8; DpK_(a)/DT=−0.021; FW 179.2),Ethanolamine (2-Aminoethanol; C₂H₇NO; pK_(a)=9.5 at 25° C.; useful pHrange 8.8-10.2; FW 61.08), CAPSO(3-[Cyclohexylamino]-2-hydroxy-1-propanesulfonic acid; C₉H₁₉NO₄S;pK_(a)=9.6 at 25° C.; useful pH range 8.9-10.3; FW 237.3), BIS-TRIS(bis[2-hydroxyethyl]iminotris-[hydroxymethyl]methane;2-bis[2-Hydroxyethyl]amino-2-[Hydroxymethyl]-1,3-propanediol; C₈H₁₉NO₅;pK_(a)=6.5 at 25° C.; useful pH range 5.8-7.2; ΔpK_(a)/ΔT=−0.020; FW209.2); ACES (2-[2-Amino-2-oxoethyl)amino]ethanesulfonic acid;N-[2-Acetamido]-2-aminoethanesulfonic acid; C₄H₁₀N₂O₄S; pK_(a)=6.8 at25° C.; useful pH range 6.1-7.5; ΔpK_(a)/ΔT=−0.021; FW 182.2).

[0017] Agarose Gel Electrophoresis

[0018] λ phage DNA/HindIII and φX174 RF DNA/HaeIII were provided by theCloning Laboratory of City of Hope. 100 bp DNA Ladder, 1 kb DNA Ladderand High Molecular Weight DNA Marker were purchased from LifeTechnologies. λ phage DNA/HindIII contains eight fragments of 125, 564,2027, 2322, 4631, 6557, 9416 and 23130 bp; φX174 RF DNA/HaeIII contains11 fragments of 72, 118, 194, 234, 271, 281, 310, 603, 872, 1078 and1353 bp; 100 bp DNA Ladder contains 16 fragments of 100, 200, 300, 400,500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500 and 2072 bp;1 kb DNA Ladder contains 23 fragments of 75, 134, 154, 201, 220, 298,344, 396, 506, 517, 1018, 1636, 2036, 3054, 4072, 5090, 6108, 7126,8144, 9162, 10180, 11198, 12216 bp; High molecular DNA marker contains13 fragments of 8271, 8612, 10086, 12220, 15004, 17057, 19399, 22621,24776, 29942, 33498, 38416, 48502 bp.

[0019] Wide mini-sub^(R) cell GT from Bio-Rad (15 cm×16 cm) was used foragarose gel electrophoresis. A horizontal slab gel (10 cm×15 cm×6.6 mm)was cast with a 20-teeth comb (each 0.75 mm×4.8 mm with 15 μl of volumecapacity). Triethanolamine/TRICINE (TRI/TRI), Ethanolamine/CAPSO(ETH/CAP), and BIS-TRIS/ACES (BIS/ACE), TAE (Tris/Acetate) and TBE(Tris/Borate) buffers were tested with 600 ml volume. Voltage, currentand power were recorded with an EC-400 power supply (E-C Apparatus). 15μl of sample was loaded and the gel was stained with ethidium bromidefor UV photography by a CCD camera (Bio-Rad Gel Doc 1000) andMulti-Analyst^(R) software (version 1.1). Regression equation andcorrelation coefficient were obtained between the relative mobility tothe 2036 bp fragment of 1 kb DNA ladder and the log (base pair). DNAfragments ranging from 1018 bp to 23130 bp in λ DNA marker/HindIII and 1kb DNA ladder were calculated on 0.6% agarose gel. The mobility wasrelative to the 600 bp fragment of 100 bp DNA ladder and DNA fragmentsranged from 72 bp to 2072 bp in φX174 DNA marker/HaeIII, 100 bp DNAladder and 1 kb DNA ladder on 2.5% agarose gels. The mobility wasrelative to the 1636 bp fragment of 1 kb DNA ladder and DNA fragmentsranged from 300 bp to 12216 bp in 100 bp DNA ladder and 1 kb DNA ladderwere calculated on 1% agarose gels.

[0020] Sequence Analysis

[0021] Exons E and H of the human factor IX gene were amplified withprimers I4(17305)-17D and I5(18163)-17U to generate on 858 bp regionincluding exon E, or with primers I7(30412)-17D and E8(31573)-17U for a1161 bp region of exon H. [Nomenclature as described (13, 14), as anexample, E8 (17305)-17D is an oligonucleotide in which the 5′ end beginsat basepair 17305. The length is 17 bases, and the orientation is“downstream” (D), i.e., in the direction of transcription.] The PCRmixture contained a volume of 25 μl; 50 mM KCl, 10 mM Tris/HCl pH 8.3,1.5 mM MgCl₂ for exon E or 2.5 mM MgCl₂ for exon H, 200 μM of each dNTP,0.1 μM of each primer, 0.5 U of Taq DNA polymerase (Boehringer Mannheim)and 250 ng of genomic DNA. Denaturation was at 94° C. for one minute,annealing was at 55° C. for one minute, and elongation was at 72° C. forthree minutes, for a total of 30 cycles. The PCR product was purified byMicrocon^(R) 100 (Amicon). Sequencing reaction was performed with ThermoSequenase radiolabeled terminator cycle sequencing kit (Amersham LifeScience) utilizing primer I5(17866)-19U for exon E or primerE8(31164)-16U for exon H. Bio-Rad-Sequi-Gen GT sequencing cell (38 cm×50cm×0.4 mm) was used. A 7M urea 6% LongRanger™ gel with 73 shark-toothlanes was electrophoresed with a total of 3000 ml buffer at 80W constantpower and 2200-2500 volts (Bio-Rad PowerPac 3000 Power Supply). TRI/TRI,ETH/CAP, and BIS/ACE buffers at 30 mM, and TBE buffer at 50 mM weretested. After a preliminary electrophoresis for 30 minutes, three μl ofeach sample was loaded and electrophoresed for two hours at 45° C. Thegel was dried and exposed to Kodak BioMax MR film for autoradiography.The sequence was read by SEQ-EASY™ digitizer-talker and DNA*™ software(DNASTAR) and was aligned with wildtype sequence.

[0022] DideoxyFingerprinting (ddF)

[0023] Samples were amplified as mentioned above, except with primersI1(6094)-30D and I3(6878)-27U for a 785 bp region of exons B/C or withprimers I7(30646)-34D and E8(31645)-31U for a one kb region of exon H inthe human factor 1× gene. The ddF reactions were identical to a singledideoxy component of the sequencing reactions, with the exception ofprimers and loading buffer: primer I1(6272)-22D for exons B/C or with[α-³³P]ddGTP and primer I7(30851)-19D for exon H and 15 μl ofstop/loading buffer (50% formamide, 7M urea, 2 mM EDTA, 0.05%bromophenol and 0.05% xylene cyanol) was added to each tube.

[0024] A non-denaturing gel (45 cm×37.5 cm×0.4 mm) was electrophoresedby using a water-cooled PokerFace™ SE 1500 sequencing apparatus withtotal 4000 ml buffer at 12-15 watts constant power for 6-16 hours.TRI/TRI and ETH/CAP buffers at 30 mM, and TBE buffer at 50 mM weretested. On the ddF gel, an informative dideoxy component was easilydetected by a missing or an extra segment. The shiftedmutation-containing segments in the SSCP component were scored bycomparison with wildtype control. Typically, a migration change of ½band width on the upper part of the gel or ¼ band width on the lowerpart was the limit of resolution (15).

RESULTS

[0025] Properties of the Buffers

[0026] As illustrated in FIG. 1A, pK-matched buffers are predicted toprovide more stable current and voltage during electrophoresis becauseboth cations and anions can be regenerated at the anode and the cathode,respectively.

[0027] Referring to FIG. 1B, these predictions were confirmed when theelectrophoretic stability of pK-matched buffers was compared with thatof TBE by performing an SSCP-type analysis for 12 hours with theALF-express fluorescent sequencer. 8% non-denaturing polyacrylamide-typegel (29.5 cm×30 cm×0.5 mm) was electrophoresed for 12 hours with theALFexpress™ DNA Sequencer (Pharmacia Biotech) with 1000 ml ofelectrophoresis buffer in each of upper and lower reservoirs. Thevoltage, current and power were set at 1500V, 40 mA and 35 watt and thegel was electrophoresed for 12 hours at 35° C. The ETH/CAP pK-matchedbuffer at 30 mM (Upper) was compared with TBE buffer at 50 mM (Lower).The Y axes represent the observed voltage (−), current (---) and power(--). The X axis is the running time. pK-matched buffer at 30 mM wasshown more stable with the voltage, current and power than the TBEbuffer at 50 mM. The same results were obtained when pK-matched bufferwas used at 50 mM and when denaturing sequencing gels were applied.

[0028] Table 1 shows the chemical properties of three pK-matched buffersof Triethanolamine/TRICINE (TRI/TRI), Ethanolamine/CAPSO (ETH/CAP), andBIS-TRIS/ACES (BIS/ACE). The two components of each buffer are a weakacid and a weak base having similar pK_(a) values (|ΔpK_(a)|≦0.3). ThepH of each buffer is close to the average of pK_(a) values with 1:1molar mixture of acid and base (|pH−½×(pK_(a)+pK_(a)′)|≦0.1). The pH ofthe buffers range from 6.7 to 9.6. The buffers had been stored for 4-12months and were chemically stable (CAPSO is light sensitive and shouldbe stored in dark). For example, 30 mM TRI/TRI buffer contains 30 mMTriethanolamine and 30 mM TRICINE (pH 7.9 at 25° C.). Table 2 summarizesthe physical properties of current, power, and voltage in agarose gelelectrophoresis. pK-matched buffer was compared with TBE and TAE atvarious concentrations from 50 mM to 5 mM (1× TAE: 40 mM Tris/26.6 mMAcetate, 1 mM EDTA, pH 8.0).

[0029] Agarose Gel Electrophoresis

[0030] Linear duplex DNA molecules migrate at a rate that is inverselyproportional to the logarithm of their molecular weight (16). DNAmarkers, which sizes ranged from 48.5 kb to 72 bp, were electrophoresedon 1% agarose gels at various buffer concentrations with constantvoltage (6 voltage/cm for 1.5 hour. The mobilities of the pK-matchedbuffers were compared with TBE and TAE buffers from 50 mM to 5 mM. Theresults are shown in FIG. 2: A. TRI/TRI, B. ETH/CAP, C. BIS/ACE, D. TBE;Lanes 1-4, 5-8, 9-12 and 13-16 were at 50, 25, 10 and 5 mM. E. TAE, at40, 20, 10 and 5 mM, respectively. DNA markers: In lanes 1, 5, 9 and 13were 500 ng of λ DNA/HinHIII; in lanes 2, 6, 10 and 14 were 500 ng ofφX174 DNA/HaeIII; in lanes 3, 7, 11 and 15 were 1000 ng of 100 bp DNAladder; in lanes, 4, 8, 12 and 16 were 1000 ng of 1 kb DNA ladder. Thetop of the image was the well position.

[0031] The relative mobilities of the DNA fragments from 100-12000 bpwere inversely proportional to the log of the sizes and the correlationcoefficients (r) were close to −1 under each condition (Table 2). Withdecreased buffer concentrations from 50 mM to 5 mM, absolute mobilitiesof the DNA fragments decreased, typically by a factor of 1.3-2.0 (FIG.2).

[0032] The broadening of bands were also analyzed. Broad bands resultfrom diffusion of small DNA molecules through the gel (2,12) and fromdispersion of large DNA molecules through entanglement (17). Thesegments of small sizes became sharper with diluted bufferconcentration, but the band of the 2072 bp fragment in the 100 bp DNAladder broadened. When the concentration was as low as 5 mM, pK-matchedbuffer still provided high resolution without smearing, while TBE or TAEbuffers did not (FIG. 2).

[0033] The pK-matched buffers were tested at 15 mM and compared with 1×TBE and 1× TAE buffers to determine the effects of agarose concentrationand applied voltage. The results are shown in FIG. 3:

[0034]FIG. 3A: 0.6% agarose gel was electrophoresed at 4 volts/cm for 3hours. DNA markers: In lane 1 was 500 ng of λ DNA/HinHIII; in lane 2 was1 μg if 100 bp DNA ladder, in lane 3 was 1 μg of 1 kb DNA ladder. The100 bp DNA ladder was electrophoresed out of the gel.

[0035]FIG. 3B: 2.5% agarose gel was electrophoresed at 6 volts/cm for 90minutes. DNA markers: in lane 1 was 1 μg of 100 bp DNA ladder; in lane 2was 1 μg of 1 kb DNA ladder.

[0036]FIG. 3C: 1% agarose gel was electrophoresed at 12 volts/cm for 45min. DNA markers were the same as in B.

[0037] The quality of separation achieved with the pK-matched buffers at15 mM was as high as or better than that achieved with 1× standardconcentrations of TBE (50 mM) or TAE (40 mM), when the effects of lowagarose gel concentrations and high voltage were examined (FIG. 3 andTable 3).

[0038] Sequence Analysis

[0039] Sanger dideoxy sequencing analyses were performed on 7M ureaLongRanger™ gels for exons H and E in the factor IX gene (7,8). TRI/TRI,ETH/CAP and BIS/ACE buffers at 30 mM were compared with 1× TBE buffer.The results are shown in FIG. 4: A. TRI/TRI buffer, B. ETH/CAP buffer,C. BIS/ACE buffer. The three pK-matched buffers were used at 30 mM. D.1× TBE buffer (50 mM) for comparison. Two regions of exons H (left) andE (right) in the factor IX gene were shown. Under each condition, thetermination segments were separated with high resolution. The sequenceswere obtained accurately up to 360 bases for exon H and up to 230 basesfor exon E, which were read to the last base by SEQ-EASY™digitizer-talker and DNA*™ software. A heterozygous mutation C-T in exonH was detected with all conditions (FIG. 4). Similar results wereobserved when other regions were sequenced (data not shown).

[0040] Dideoxy Fingerprinting

[0041] Dideoxy fingerprinting (ddF) was used to efficiently explore theSSCP sensitivity of different conditions. ddF is a hybrid techniquebetween SSCP and Sanger dideoxy sequencing (9, 10, 15). ddF reaction isperformed with one primer and one dideoxy terminator; then theterminated single-stranded segments are electrophoresed through one laneof a non-denaturing gel. The ladder of segments subsequent to a mutationconstitutes the SSCP component, which contain the same mutation butdiffer at the 3′ ends.

[0042] Nondenaturing conformation-sensitive electrophoresis of markedlydifferent sized segments such as that performed with dideoxyfingerprinting, pose a challenge for electrophoresis buffers. SometimesTBE buffer must be changed during electrophoresis in order to maintainresolution.

[0043] TRI/TRI and ETH/CAP buffer were compared with TBE buffer. Theresults for TRI/TRI and TBE are shown in Fig. SA and 5B, respectively.ddF gels were performed with (A) splice 30 mM TRI/TRI and (B) 1× TBE forexons B/C. The segments up to 300 bases were scored for the first 22mutations, in which the number of the mutation-containing segmentsvaried from 15 to 26, depending on the location of the mutation. Theaverage efficiencies of SSCP component were 71% for both the conditions,but variable efficiencies of an individual mutation varied. Theefficiencies between the two conditions for each mutation werecorrelated (coefficient=0.88).

[0044] 10% PAGE^(plus) gels were electrophoresed at 20° C. Lanes 1-28were hemizygous for the following mutations in exons B/C. 1:C6364T;2:G6365T; 3:G6365A; 4:G6374A; 5:G6375T; 6:G6376C; 7:A6379G; 8:G6385A;9:G6394A; 10:A6398G; 11:T6401C; 12:G6436A; 13:T6442C; 14:G6451C;15:G6454A; 16:C6460T; 17:G6461A; 18:G6461C; 19:G6463A; 20:G6463C;21:C6488T; 22:T6495C; 23:C6575G; 24:A6653G; 25:G6677C; 26:A6690T;27:T6696G; 28:A6693G. Lane C was wildtype control.

[0045] Other electrophoresis conditions were also tested, which includedgel matrices of MDE (FMC BoiProducts), HR1000 (Genomyx), PAGE^(plus)(Amresco), and Dcode™ (Bio-Rad); additives of glycerol, urea, ResolverGold™ and PEG; and temperatures at 20° C. and 8° C. Twenty-twoelectrophoretic conditions were tested with different combinations ofthe buffers, gel matrices, additives, and temperatures. The pK-matchedbuffers gave sharp bands with resolution at least equivalent to that ofTBE (FIG. 5B).

[0046] Other PK-Matched Buffers

[0047] Two other pK-matched buffers of 2-Amino-2-methyl-1-propanol(pK_(a)9.7)/CAPSO (pK_(a)9.6) with pH 9.6 at 25° C., and Triethanolamine(pK_(a)7.8)/HEPES (N-[2-Hydroxyethyl)]piperazine-N′-[2-ethanesulfonicacid, pK_(a)7.5) with pH 7.6⁺ at 25° C. were also tested and similarresults were observed (data not shown).

[0048] The pK-matched buffers in the present study were chosen such thatthe pK_(a) of the acid and the pK_(a)′ of the base were within 0.3 unitsof one another. In each of the pK-matched buffers, the ionic strength(M) of each component is ≦50% of its molar concentration, because pK_(a)of the acid is ≧ pK_(a)′ of the base, which contributes to theelectrophoretic stability (Table 1). The ionic strength of eachcomponent is calculated from Henderson-Hasselbalch equation to be 12.4mM to 14.2 mM in 30 mM pK-matched buffers. Each of [Tris⁺] and[Acetate⁻] is 26.6 mM in 40 mM TAE buffer (pH 8.0); [Tris⁺] is 25.0 mMin 50 mM TBE (pH 8.3) with Borate⁻ complex formation. TRI-TRI buffer isparticularly advantageous for routine analysis in molecular biologylaboratories, since 200× stocks can conveniently be produced.

[0049] In summary, pK-matching buffers were developed and tested onagarose gels for separation of double-stranded DNA segments, ondenaturing polyacrylamide gels for sequencing analysis, and onnon-denaturing gels for SSCP analysis. High electrophoretic stabilityand high resolution were observed even at low working concentrations.

[0050] In the experiments, parameters, such as gel concentration,chamber capacity, voltage and power, were set as the optimal for TBE andTAE buffers, so the pK-matched buffers should be even better with theiroptimal conditions. Because of the higher electrophoretic stability of apK-matched buffer, the reservoirs of electrophroesis cells may beeliminated, or the volume of the reservoirs may be greatly reduced, to50% or less of the volume of reservoirs in electrophoresis cells usingTAE or TBE buffers, especially for agarose gel electrophoresis. Forexample, a Bio-Rad cell used for agarose gel electrophoresis has tworeservoirs with a total volume of 600 μl. These can be eliminated orreduced to a total volume less than 300 μl for use with pK-matchedbuffers of this invention.

[0051] Our invention is not limited to the particular pK-matched buffersused on our experiments. The invention includes buffers prepared bymixing any weak acid and any weak base having pKa values within about0.3 units of one another. pK-matched buffers can be generated for anydesired pH value, so long as appropriate acids and bases are available.pK-matched buffers are useful for electrophoresis analysis of proteinsand small molecules.

[0052] A variety of other buffers have been used for electrophoresis:(3,18-20), e.g., Tris/phosphate (1× TPE: 90 mM Tris/28 mM phosphoricacid/2 mM EDTA), Alkaline “running buffer” (1×: 50 mM NaOH, 1 mM EDTA,pH 12-13), Tris/glycine (1×: 25 mM Tris/250 mM glycine, 0.1% SDS, pH 8.3for SDS-polyacrylamide gel), Tris/Taurine (1×: 89 mM Tris/29 mMTaurine/0.5 mM EDTA, pH 9.0) (21), Barbitone/acetate (pH 8.6, a standardbuffer for immunoelectrophoresis and separation of serum protein) (22)and Non-barbitone buffer (23), and some other buffers (24-28). Theseabove buffers either contain only one effective component or two moreeffective components without pK matching. TABLE 1 Chemical properties ofpK-matched buffers Second First Component Component Stock Buffer^(a)Base pK_(a) ^(l) Acid pK_(a) pH^(b) Mole Ratio Conc. (M) TRI/TRITriethanolamine 7.8 TRICINE 8.1 7.9 1:1 3 ETH/CAP Ethanolamine 9.5 CAPSO9.6 9.6 1:1 0.5 BIS/ACE BIS-TRIS 6.5 ACES 6.8 6.7 1:1 0.5

[0053] TABLE 2 Comparison on agarose gel^(a) Separation PhysicalProperty Correlation Power Current Regression Coefficient Buffer Conc.(mM) (Watt) (mA) Heating Equation^(b) (r)^(b) TRI/TRI 50 6.5 70 + y =−1.30x + 4.55 −0.992 25 3.2 40 No y = −1.29x + 4.53 −0.992 10 1.2 16.5No y = −1.34x + 4.42 −0.980 5 0.6 9.5 No y = −1.33x + 4.14 −0.942ETH/CAP 50 9.3 90 + y = −1.24x + 4.48 −0.993 25 4.5 50 No y = −1.16x +4.43 −0.992 10 1.8 23 No y = −1.27x + 4.38 −0.979 5 0.9 13 No y =−1.32x + 4.13 −0.939 BIS/ACE 50 7.0 80 No y = −1.21x + 4.47 −0.993 253.5 43 No y = −1.23x + 4.49 −0.992 10 1.5 19 No y = −1.29x + 4.38 −0.9825 0.7 9.5 No y = −0.97x + 4.08 −0.971 TBE 50 4.5 50 No y = −1.09x + 4.38−0.988 25 2.7 32 No y = −1.32x + 4.443 −0.978 10 1.2 13 No y = −1.03x +4.37 −0.967 5 0.6 8 No y = −1.14x + 3.99 −0.914 TAE 40 10.5 112 ++ y =−1.33x + 4.57 −0.996 20 5.5 62 + y = −1.50x + 4.73 −0.992 10 2.5 30 No y= −1.33x + 4.61 −0.988 5 1.5 18 No y = −1.33x + 4.61 −0.988

[0054] TABLE 3 Test of pK-matched buffers on agarose gel 0.6% Agarose at2.5% Agarose at 4 volts/cm^(a) 6 volts/cm^(b) 1% agarose at 12volts/cm^(c) Conc. Power Current Power Current Power Current Buffer (mM)(Watt) (mA) (Watt) (mA) (Watt) (mA) Heat TRI/TRI 30 1.8 31 3.5 43 19.5 →28^(e) 100 → 140^(e) + 15 0.9 16 1.8 22   10 → 11.5  55 → 65 No ETH/CAP30 1.8 34 5.5 63   26 → 35 130 → 180 ++ 15 0.9 17 2.5 34 13.5 → 16  70 →80 + BIS/ACE 30 1.8 34 4 50  22  110 + 15 0.9 17 2 25  10   55 No TBE 502.5 37 4.5 50   22 → 30 105 → 150 + 30 1.3 27 3.5 46  7.8 → 8.0  65 → 70No 15 0.7 16 2 24   4   37 No TAE^(d) 40 4.5 78 10.5 112 >>35^(f)>>200^(f) +++ 30 3.6 65 9 100 17.2 → 22.5 137 → 168 ++ 15 1.9 35 4.5 50  8 → 8.7  68 → 77 No

REFERENCES

[0055] 1. Tiselius, A. (1937) Annu. Rev. Med., 48, 231-240.

[0056] 2. Voytas, D. (1988) in Current Protocols in Molecular Biology(Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J.G., Smith, J. A., and Struhl, K. Eds.), pp. 2.5.1-2.5.9, John Wiley &Sons, Brooklyn, N.Y.

[0057] 3. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) inMolecular Cloning: A Laboratory Manual. pp. 6.1-6.62, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.

[0058] 4. McDonell, M. W., Simon, M. N., and Studier, F. W. (1977) J.Mol. Biol. 110, 119-146.

[0059] 5. Southern, E. (1979) Meth. Enzymol. 68, 152-176.

[0060] 6. Schwartz, D. C. and Cantor, C. R. (1984) Cell 37, 67-76.

[0061] 7. Sanger, F., Nichlen, S. and Coulson, A. R. (1977) Proc. Natl.Acad. Sci. U.S.A. 75, 5463-5467.

[0062] 8. Innis, M. A., Myambo, K. B., Gelfand, D. H., and Brow, M. A.D. (1988) Proc. Natl. Acad. Sci. USA 85, 9436-9440.

[0063] 9. Orita, M., Iwahana, H., Kanazawa, H., Hayashi, K., and Sekiya,T. (1989) Proc. Natl. Acad. Sci. USA 86, 2766-2770.

[0064] 10. Sarkar, G., Yoon, H. and Sommer, S. S. (1992) Genomics 13,441-443.

[0065] 11. Liu, Q. and Sommer, S. S. (1995) BioTechniques 18, 470-477.

[0066] 12. Kuhn, R. and Hoffstetter-Kuhn, S. (1993) in CapillaryElectrophoresis: Principles and Practice. pp. 37-101, Springer-Verlag,New York, N.Y.

[0067] 13. Yoshitake, S., Schach, B. G., Foster, D. C., Davie, E. W.,and Kurachi, K. (1985) Biochemistry 24, 3736-3750.

[0068] 14. Sarkar, G. and Sommer, S. S. (1989) Science 244, 331-334.

[0069] 15. Liu, Q. and Sommer, S. S. (1994) PCR Methods and Applications4, 97-108.

[0070] 16. Helling, R. B., Goodman, H. M., and Boyer, H. W. (1974) J.Virol. 14, 1235-1244.

[0071] 17. Yarmola, E., Sokoloff, H. and Chramback, A. (1996)Electrophoresis 17, 1416-1419.

[0072] 18. Good, N. E., Winget, G. D., Winter, W., Connolly, T. N.,Izawa, S. and Singh, R. M. (1966) Biochemistry 5, 467-477.

[0073] 19. Stoll, V. S. and Blanchard, J. S. (1990) Methods Enzymol.182, 24-38.

[0074] 20. Ellis, K. J. and Morrison, J. F. (1982) Methods Enzymol. 87,405-26.

[0075] 21. Ganguly, A., Rock, M. J. and Prockop, D. J. (1993) Proc.Natl. Acad. Sci. U.S.A. 90, 10325-10329.

[0076] 22. Kohn, J. and Riches, P. G. (1978) J. Immunol. Methods 20,325-331.

[0077] 23. Ambler, J. and Rodgers, M. (1980) Clin. Chem. 26, 1221-23.

[0078] 24. Liu, Q. and Sommer, S. S. (1998) BioTechniques 25, 50-56.

[0079] 25. Kukita, Y., Tahira, T., Sommer, S. S., and Hayashi, K. (1997)Hum. Mutat. 10, 400-07.

[0080] 26. Sasaki, T., Tian, H., Kukita, Y., Inazuka, M., Tahira, T.,Imai, T., Yamauchi, M., Saito, T., Hori, T., Hashimoto-Tamaoki, T.,Komatsu, K., Nikaido, O., and Hayashi, K. (1998) Hum. Mutat. 12,186-195.

[0081] 27. Orban, L., Tietz, D. and Chramback, A. (1987) Electrophoresis8, 465-471.

[0082] 28. Chramback, A. and Jovin, T. M. (1983) Electrophoresis 4,190-200.

In the claims:
 1. A pK-matched buffer comprising a weak acid and a weakbase which have pK_(a) values at 25° C. within about 0.3 units of oneanother.
 2. A pK-matched buffer of claim 1, wherein the weak acid isCAPSO and the weak base is ethanolamine.
 3. A pK-matched buffer of claim1, wherein the weak acid is ACES and the weak base is BIS-TRIS.
 4. ApK-matched buffer of claim 1, wherein the weak acid is CAPSO and theweak base is 2-amino-2-methyl-1-propanol.
 5. A pK-matched buffer ofclaim 1, wherein the weak acid is HEPES and the weak base istriethanolamine.
 6. In an electrophoresis method, the improvement whichcomprises running the electrophoresis in a pK-matched buffer comprisinga weak acid and a weak base which have pK_(a) values at 25° C. withinabout 0.3 units of one another.
 7. The method of claim 6, wherein thepK-matched buffer is selected from a buffer comprising CAPSO as the weakacid and ethanolamine as the weak base; a buffer comprising ACES as theweak acid and BIS-TRIS as the weak base; a buffer comprising CAPSO asthe weak acid and 2-amino-2-methyl-1-propanol as the weak base; and abuffer comprising HEPES as the weak acid and triethanolamine as the weakbase.
 8. The method of claim 6, wherein the method is a gelelectrophoresis method for separating nucleic acids or polypeptides. 9.The method of claim 7, wherein the method is a gel electrophoresismethod for separating nucleic acids or polypeptides.
 10. The method ofclaim 7, wherein the pk-matched buffer is a buffer comprising CAPSO asthe weak acid and ethanolamine as the weak base.
 11. The method of claim7, wherein the pK-matched buffer is a buffer comprising ACES as the weakacid and BIS-TRIS as the weak base.
 12. The method of claim 7, whereinthe pK-matched buffer is a buffer comprising CAPSO as the weak acid and2-amino-2-methyl-1-propanol as the weak base.
 13. The method of claim 7,wherein the pK-matched buffer is a buffer comprising HEPES as the weakacid and triethanolamine as the weak base.